Urothelial cell platelet-activating factor production mediated by calcium-independent phospholipase A₂γ

Objectives: To determine the effect of phospholipase A₂ (PLA₂) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation. Methods: Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA₂ activity in TEU-2 cells was measured using (16:0, [³H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [ ³H]PAF production was measured in TEU-2 cells prelabeled with [ ³H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [³H] acetate hydrolyzed from [ ³H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells. Results: Most PLA₂ activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA₂ and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA₂γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA₂ inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition. Conclusions: Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA₂γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.